Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1996-05-09
2000-10-31
Achutamurthy, Ponnathapura
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435 691, 435212, 4352523, 435471, 536 232, 530350, 5303911, 5303917, 424943, 4241341, C12N 964, C12N 1557, C12N 1562, C12N 1579
Patent
active
061401005
DESCRIPTION:
BRIEF SUMMARY
This application is filed pursuant to 35 U.S.C. .sctn.371 as a United States National Phase Application of International Application No. PCT/GB94/02483 filed 11 Nov. 11, 1994 which claims priority from GB 9323429.2 filed Nov. 12, 1993.
FIELD OF THE INVENTION
The present invention relates to improvements in targetted enzyme prodrug therapy including antibody-directed enzyme prodrug therapy (ADEPT), it particularly relates to novel enzymes and prodrugs for use in ADEPT.
In the therapy of certain conditions it is preferable that a drug be delivered onto to a particular cellular subpopulation. For example the use of drugs in the treatment of cancer is limited by the inability of the cytotoxic drug to differentiate between cells exhibiting normal cell division and those exhibiting neoplastic division. Hence the therapy is not targeted to a clinically acceptable extent and healthy cells are exposed to cytotoxin. Conjugation of a drug to an antibody, preferably a monoclonal antibody (mAb), allows the targeting of the drug to a particular cellular subpopulation expressing the antigenic determinant to which the targetting antibody binds. However, factors, such as the inability of the conjugate to penetrate the relevant tissue, poor release of the drug from the antigen bound conjugate and the limitation placed on the amount of drug which can be delivered by the number of available antibody-binding sites, have limited the effectiveness of this approach.
BACKGROUND
Avoidance of such limitations led to the concept of targeting conjugates of antibodies and enzymes capable of converting relatively non-cytotoxic `prodrugs` into low molecular weight cytotoxins at the antibody-binding site. This general concept was disclosed by Rose in European Patent application 84302218.7. Bagshawe and collaborators have referred to the concept as ADEPT, Antibody Directed Enzyme Prodrug Therapy. (Bagshawe K. D. et.al., Br. J. Cancer [1987] 56, 531-532, Bagshawe K. D. et al., Br. J. Cancer [1988] 50, 700-703 and WO 90/10140). In this way one conjugate could generate a proportionately larger amount of drug at the target site by repeated rounds of enzymatic catalysis of prodrug activation.
EP 382 411 describes ADEPT wherein a prodrug may be converted to a cytotoxic agent by enzymes including beta-lactamases isolated from various micro-organisms, L-pyroglutamate aminopeptidase, beta-galactosidase, D-amino acid peptidase, isoenzymes of alkaline phosphatase and various carboxypeptidases.
WO 91/11201 describes ADEPT wherein a prodrug may be enzymatically cleaved to generate cyanide by .beta.-glycosidases or .beta.-glucosidases, generally of plant origin.
EP 302 473 describes ADEPT wherein the enzyme alkaline phosphatase may be used to cleave novel prodrugs of mitomycin, penicillin V amidase may be used to cleave novel prodrugs of adriamycin, or cytosine deaminase may be used with the prodrug 5-fluorocytosine.
EP 484 870 describes ADEPT wherein .beta.-lactamase may be used to activate a cephalosporin prodrug to yield a cytotoxic nitrogen mustard.
WO 88/07378 describes ADEPT wherein benzoic acid nitrogen mustard glutamides may be converted to the nitrogen mustard under the action of carboxypeptidases.
Vitols, K. S., et al, Pteridines 3, [1992], 125-126, discloses a number of MTX-amino acid prodrugs which may be activated by carboxypeptidase-mAb conjugates as part of ADEPT.
WO 91/09134 discloses bispecific hybrid mAbs for use in ADEPT wherein the mAb has specificities against both human cancer cell antigens and a prodrug-activating enzyme.
All previously disclosed methods of ADEPT may be divided into two categories: those which employ human enzymes and those which employ non-human (eg. bacterial) enzymes to activate the relevant prodrug. Both strategies retain inherent problems which limit their potential to provide effective therapy. Use of a human enzyme results in instability of the associated prodrug in vivo, as it may be activated at sites distant to the target site where endogenous human enzymatic activity may occur naturally. Clearly t
REFERENCES:
Olesen K. et al., "Altering Substrate Preference of Carboxypeptidase Y by a Novel Strategy of Mutagenesis Eliminating Wild Type Background", Protein Engineering, England, GB, 6(4), 409-415 (1993).
Phillips, Margaret A. et al., "Transition State Characterization: A New Approach Combining Inhibitor Analogues and Variation in Enzyme Structure", Biochemistry, Easton, PA, US, 31, 959-963, (1992).
Kuefner, Ulrike et al., "Carboxypeptidase-Mediated Release of Methotrezate From Methotrexate Alpha-Peptides", Biochemistry, Easton, PA, US, 28, 2288-2297 (1989).
Blumenkopf Todd Andrew
Cory Michael
Smith Gary Keith
Achutamurthy Ponnathapura
Bennett Virginia C.
Glaxo Wellcome Inc.
Grassler Frank P.
Hrubiec Robert T.
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